1. Field of the Invention
The present invention relates to compositions comprising ultra-pure complexes of insulin-like growth factor I and insulin like growth factor binding protein-3, and methods of making the same.
2. Description of the Related Art
IGF-I/IGFBP-3 is a protein complex of insulin-like growth factor-I (“IGF-I”) and insulin like growth factor binding protein-3 (“IGFBP-3”). IGF-I is a small polypeptide with strong structural and functional homology to pro-insulin. As such, IGF-I elicits many of the physiological effects of insulin.
IGF-I/IGFBP-3 complexes may be used for the treatment of a wide array of disorders (See, e.g., U.S. Pat. Nos. 5,681,818, 5,723,441, 5,948,757, 6,015,786, 6,017,885, 6,025,332, 6,025,368, 6,514,937, and 6,518,238). In healthy individuals, IGF-I can be found within the blood circulation bound by other proteins. For example, IGF-I is frequently bound to IGFBP-3, the most abundant IGF-I binding protein. The IGF-I/IGFBP-3 complex associates with and an acid-liable subunit protein, forming a 150 kD complex. See Adams et al., Prog. Growth Factor Res. 6(2-4):347-56 (1995). This large ternary complex serves as a circulatory reservoir of IGF-I as IGF-I/IGFBP-3 complexes exhibit a longer half-life and improved stability as compared to free IGF-I. See Adams et al., supra, and Blum et al. (1991), Plasma IGFBP-3 Levels as Clinical Indicators, in Modern Concepts of Insulin-like Growth Factors, pp. 381-93, E. M. Spencer, ed., Elsevier, N.Y.
IGF-I, IGFBP-3, and IGF-I/IGFBP-3 complexes can be obtained from natural sources or by recombinant techniques. Recombinant technology can be used to produce IGF-I, IGFBP-3, and IGF-I/IGFBP-3 complexes in eukaryotic and prokaryotic organisms (See, e.g., U.S. Pat. Nos. 5,200,509, 5,670,341, 5,789,547, and 6,417,330). Recombinant IGF-I, IGFBP-3, and IGF-I/IGFBP-3 complexes can be cultured in batch or continuous formats, with the harvesting of either the cell culture supernatant or the recombinant cells themselves.
IGF-I, IGFBP-3, and IGF-I/IGFBP-3 complexes typically are purified after expression in recombinant systems using such techniques as size exclusion chromatography, hydrophobic interaction chromatography, and ion exchange chromatography. However, such techniques fail to remove all impurities. For example, IGF-I/IGFBP-3 complexes typically are present in partially purified preparations containing protein aggregates. Moreover, new impurities, such as mass and charge variants of IGFBP-3, have been discovered that are not removed by prior art techniques. FIG. 1 provides a cation exchange trace obtained from the linear gradient carboxymethyl ion exchange (“CM-IEX”) chromatography of samples comprising IGF-I/IFGBP-3 complexes and protein aggregates. FIG. 2 provides a LC/MS analysis of IGF-I/IGFBP-3 complexes purified using linear gradient CM-IEX showing newly discovered mass and charge variants.
It is well accepted in the Pharmaceutical arts that drug purity is highly desired and that even small improvements in drug purity are important improvements. This is due to the fact that impurities may have unanticipated impact on drug stability, safety, or efficacy. Accordingly, improved methods of purifying IGF-I/IGFBP-3 complexes are inherently useful and needed.